Sequencing of the human genome, and the genomes of other species, has emphasized the fact that the expression and properties of a protein are often dependent on posttranslational modifications and, thus, cannot be predicted from the DNA sequence. This realization has spurred an interest in proteomics, the study of protein expression within a cell under defined conditions.
Traditionally, proteins from biological samples have been isolated and identified by separating the proteins using 2-D gel electrophoresis followed by identification of the protein using mass spectrometry. However, this method is time consuming and can only detect proteins that are highly abundant in the biological sample. Severe streaking causes deterioration in resolution of the electrophoretic separation when high loading is used in an attempt to visualize less abundant proteins.
Particular difficulties have been encountered in attempts to use 2-D gel electrophoresis/mass spectrometry to study phosphorylated proteins or peptides, as they are often present in low abundance. Such proteins and peptides are of particular interest in proteomic studies, however, as signal transduction and other cellular processes are often regulated by phosphorylation/dephosphorylation cascades. In fact, approximately one-third of all proteins expressed by mammalian cells contain covalently bound phosphate groups.
One method currently in use for selectively enriching phosphorylated proteins in a sample involves β-elimination of the phosphate group from a phosphoserine or phosphothreonine amino acid residue to form an α,β-unsaturated carbonyl. The α,β-unsaturated carbonyl is a Michael acceptor and can react with a nucleophilic linker, such as ethanedithiol, which can be used to link the protein to a biotin affinity tag. Proteins having the biotin affinity tag are then selectively separated. However, this method suffers from the disadvantage that it cannot detect proteins that have phosphorylated tyrosine residues. In addition, a Michael reaction with a nucleophilic linker generates diastereoisomers which can complicate the analysis of the sample.
A second method for enriching the abundance of phosphorylated proteins in a sample involves selective immobilization of phosphorylated proteins on a metal-affinity column. Selectivity of this method is greatly increased when the carboxylic acid terminal of the proteins are esterified before sample is passed through the affinity column. However, since phosphorylated proteins are not covalently attached to the metal-affinity column, stringent washing conditions cannot be used to remove unwanted non-covalently associated molecules, such as cysteinyl peptides.
The above enrichment methods have greatly facilitated the study of phosphoproteomic. However, development of a phosphoprotein enrichment method that does not suffer from the disadvantages of the above methods would be desirable.